Bis-(2-aryl) hydrazones

ABSTRACT

The present invention relates to compositions and methods for inhibiting nonenzymatic cross-linking (protein aging). Accordingly, a composition is disclosed which comprises a bis-(2-aryl)hydrazone capable of inhibiting the formation of advanced glycosylation endproducts of target proteins. The method comprises contacting the target protein with the composition. Both industrial and therapeutic applications for the invention are envisioned, as food spoilage and animal protein aging can be treated.

This application is a continuation-in-part of application Ser. No.08/372,672, filed Jan. 13, 1995.

BACKGROUND OF THE INVENTION

The present invention relates generally to the aging of proteinsresulting from their reaction with glucose and other reducing sugars,and more particularly to the inhibition of the reaction ofnonenzymatically glycosylated proteins and the often resultant formationof advanced glycosylation (glycation) endproducts and cross-links.

The reaction between glucose and proteins has been known for some time.Its earliest manifestation was in the appearance of brown pigmentsduring the cooking of food, which was identified by Maillard in 1912,who observed that glucose or other reducing sugars react with aminoacids to form adducts that undergo a series of dehydrations andrearrangements to form stable brown pigments. Further studies havesuggested that stored and heat treated foods undergo nonenzymaticbrowning as a result of the reaction between glucose and the polypeptidechain, and that the proteins are resultingly cross-linked andcorrespondingly exhibit decreased bioavailability.

This reaction between reducing sugars and food proteins was found tohave its parallel in vivo. Thus, the nonenzymatic reaction betweenglucose and the free amino groups on proteins to form a stable,1-deoxyketosyl adduct, known as the Amadori product, has been shown tooccur with hemoglobin, wherein a rearrangement of the amino terminal ofthe beta-chain of hemoglobin by reaction with glucose, forms the adductknown as hemoglobin A_(1c). The reaction has also been found to occurwith a variety of other body proteins, such as lens crystallins,collagen and nerve proteins. See Bucala et al., "Advanced Glycosylation;Chemistry, Biology, and Implications for Diabetes and Aging" in Advancesin Pharmacology, Vol. 23, pp. 1-34, Academic Press (1992).

Moreover, brown pigments with spectral and fluorescent propertiessimilar to those of late-stage Maillard products have also been observedin vivo in association with several long-lived proteins, such as lensproteins and collagen from aged individuals. An age-related linearincrease in pigment was observed in human dura collagen between the agesof 20 to 90 years. Interestingly, the aging of collagen can be mimickedin vitro by the cross-linking induced by glucose; and the capture ofother proteins and the formation of adducts by collagen, also noted, istheorized to occur by a cross-linking reaction, and is believed toaccount for the observed accumulation of albumin and antibodies inkidney basement membrane.

In U.S. Pat. No. 4,758,583, a method and associated agents weredisclosed that served to inhibit the formation of advanced glycosylationendproducts by reacting with an early glycosylation product that resultsfrom the original reaction between the target protein and glucose.Accordingly, inhibition was postulated to take place as the reactionbetween the inhibitor and the early glycosylation product appeared tointerrupt the subsequent reaction of the glycosylated protein withadditional protein material to form the cross-linked late-stage product.One of the agents identified as an inhibitor was aminoguanidine, and theresults of further testing have borne out its efficacy in this regard.

While the success that has been achieved with aminoguanidine and similarcompounds is promising, a need continues to exist to identify anddevelop additional inhibitors that broaden the availability and perhapsthe scope of this potential activity and its diagnostic and therapeuticutility.

SUMMARY OF THE INVENTION

In accordance with the present invention, a method and compositions aredisclosed for the inhibition of the advanced glycosylation of proteins(protein aging). In particular, the compositions comprise agents forinhibiting nonenzymatic cross-linking (protein aging) due to theformation of advanced glycosylation (glycation) endproducts. The agentsmay be selected from those materials capable of reacting with an earlyglycosylation product from the reaction of glucose with proteins andpreventing further reactions. Cross-linking caused by other reactivesugars present in vivo or in foodstuffs, including ribose, galactose andfructose would also be prevented by the methods and compositions of thepresent invention.

The agents comprise bis-(2-aryl)hydrazone compounds having the followingstructural formula: ##STR1## wherein R is a pyridyl, phenyl or acarboxylic acid substituted phenyl group of the formula ##STR2## whereinR₃ is hydrogen, lower alkyl, W is a carbon-carbon bond or an alkylenegroup of 1-3 carbon atoms, and R₁ is a lower alkyl, aryl, or heteroarylgroup and R₂ is hydrogen, a lower alkyl, aryl or heteroaryl group;

and their biologically or pharmaceutically acceptable acid additionsalts; and mixtures thereof, and a carrier therefor.

The compounds, and their compositions, utilized in this invention appearto react with an early glycosylation product thereby preventing the samefrom later forming the advanced glycosylation end products which lead toprotein cross-links, and thereby, to protein aging.

The present invention also relates to a method for inhibiting proteinaging by contacting the initially glycosylated protein at the stage ofthe early glycosylation product with a quantity of one or more of theagents of the present invention, or a composition containing the same.In the instance where the present method has industrial application, oneor more of the agents may be applied to the proteins in question, eitherby introduction into a mixture of the same in the instance of a proteinextract, or by application or introduction into foodstuffs containingthe protein or proteins, all to prevent premature aging and spoilage ofthe particular foodstuffs.

The ability to inhibit the formation of advanced glycosylationendproducts carries with it significant implications in all applicationswhere protein aging is a serious detriment. Thus, in the area of foodtechnology, the retardation of food spoilage would confer an obviouseconomic and social benefit by making certain foods of marginalstability less perishable and therefore more available for consumers.Spoilage would be reduced as would the expense of inspection, removal,and replacement, and the extended availability of the foods could aid instabilizing their price in the marketplace. Similarly, in otherindustrial applications where the perishability of proteins is aproblem, the admixture of the agents of the present invention incompositions containing such proteins would facilitate the extendeduseful life of the same. Presently used food preservatives anddiscoloration preventatives such as sulfur dioxide, known to causetoxicity including allergy and asthma in animals, can be replaced withcompounds such as those described herein.

The present method has particular therapeutic application as theMaillard process acutely affects several of the significant proteinmasses in the body, among them collagen, elastin, lens proteins, and thekidney glomerular basement membranes. These proteins deteriorate bothwith age (hence the application of the term "protein aging") and as aconsequence of diabetes. Accordingly, the ability to either retard orsubstantially inhibit the formation of advanced glycosylationendproducts carries the promise of treatment for diabetes and, ofcourse, improving the quality and, perhaps, duration of animal life.

The present agents are also useful in the area of personal appearanceand hygiene, as they prevent the staining of teeth by cationicanti-microbial agents with anti-plaque properties, such aschlorhexidine.

Accordingly, it is a principal object of the present invention toprovide a method for inhibiting the extensive cross-linking of proteinsthat occurs as an ultimate consequence of the reaction of the proteinswith glucose and other reactive sugars, by correspondingly inhibitingthe formation of advanced glycosylation endproducts.

It is a further object of the present invention to provide a method asaforesaid which is characterized by a reaction with an initiallyglycosylated protein identified as an early glycosylation product.

It is a further object of the present invention to provide a method asaforesaid which prevents the rearrangement and cross-linking of the saidearly glycosylation products to form the said advanced glycosylationendproducts.

It is a yet further object of the present invention to provide agentscapable of participating in the reaction with the said earlyglycosylation products in the method as aforesaid.

It is a still further object of the present invention to providetherapeutic methods of treating the adverse consequences of proteinaging by resort to the aforesaid method and agents.

It is a still further object of the present invention to provide amethod of inhibiting the discoloration of teeth by resort to theaforesaid method and agents.

It is a still further object of the present invention to providecompositions including pharmaceutical compositions, all incorporatingthe agents of the present invention.

It is still further object of the present invention to provide novelcompounds, as well as processes for their preparation, for use in themethods and compositions of the present invention.

Other objects and advantages will become apparent to those skilled inthe art from a consideration of the ensuing description.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In accordance with the present invention, agents, compositions includingpharmaceutical compositions containing said agents and associatedmethods have been developed which are believed to inhibit the formationof advanced glycosylation endproducts in a number of target proteinsexisting in both animals and plant material. In particular, theinvention relates to a composition which may contain one or more agentscomprising bis-(aryl)hydrazone compounds having the structural formula:##STR3## wherein R is a pyridyl, phenyl or a carboxylic acid substitutedphenyl group of the formula ##STR4## wherein R is hydrogen, lower alkylor a water-solubilizing ester moiety; W is a carbon-carbon bond or analkylene group of 1-3 carbon atoms, and R₁ is a lower alkyl, aryl, orheteroaryl group and R₂ is hydrogen, a lower alkyl, aryl or heteroarylgroup;

and their biologically or pharmaceutically acceptable acid additionsalts; and mixtures thereof, and a carrier therefor.

The lower alkyl groups referred to above preferably contain 1-6 carbonatoms and include methyl, ethyl, propyl, butyl, pentyl, hexyl, and thecorresponding branched-chain isomers thereof. These groups areoptionally substituted by one or more halo, hydroxy, amino or loweralkylamino groups.

The alkylene groups referred to herein likewise can be straight orbranched chain, and are thus exemplified by ethylene, propylene,butylene, pentylene, hexylene, and their corresponding branched chainisomers.

In the R groups which are a carboxylic acid substituted phenyl group ofthe formula ##STR5## wherein R is hydrogen, lower alkyl or awater-solubilizing ester moiety, the water solubilizing ester moiety canbe selected from a variety of such esters known in the art. Typically,these esters are derived from dialkylene or trialkylene glycols orethers thereof, dihydroxyalkyl groups, arylalkyl group, e.g.,nitrophenylalkyl and pyridylalkyl groups, and carboxylic acid esters andphosphoric acid esters of hydroxy and carboxy-substituted alkyl groups.Particularly preferred water-solubilizing ester moieties are thosederived from 2,3-dihydroxypropane, and 2-hydroxyethylphosphate.

The aryl groups encompassed by the above formula are those containing6-10 carbon atoms, such as phenyl and lower alkyl substituted-phenyl,e.g. tolyl and xylyl, and are optionally substituted by 1-2 halo, nitro,hydroxy or lower alkoxy groups.

Where the possibility exists for substitution of a phenyl or aryl ring,the position of the substituents may be ortho, meta, or para to thepoint of attachment of the phenyl or aryl ring to the nitrogen of thehydrazine group.

The halo atoms in the above formula may be fluoro, chloro, bromo oriodo. The lower alkoxy groups contain 1-6, and preferably 1-3, carbonatoms and are illustrated by methoxy, ethoxy, n-propoxy, isopropoxy andthe like.

The heteroaryl groups referred to in the above formula contain 1-2heteroatoms, i.e. nitrogen, oxygen or sulfur, and are exemplified by befuryl, pyrrolinyl, pyridyl, pyrimidinyl, thienyl, quinolyl, and thecorresponding alkyl substituted compounds.

For the purposes of this invention equivalent to the compounds offormula (I) are the biologically and pharmaceutically acceptable acidaddition salts thereof. Such acid addition salts may be derived from avariety of organic and inorganic acids such as sulfuric, phosphoric,hydrochloric, hydrobromic, sulfamic, citric, lactic, maleic, succinic,tartaric, cinnamic, acetic, benzoic, gluconic, ascorbic, methanesulfonicand related acids.

Of the compounds encompassed by Formula I, certain substituents arepreferred. For instance, the compounds wherein W is a carbon-carbonbond, R₁ is a methyl group and R₂ is hydrogen are preferred.

Representative compounds of the present invention are:

methyl glyoxal bis-(2-hydrazino-benzoic acid)hydrazone;

methyl glyoxal bis-(dimethyl 2-hydrazinobenzoate)hydrazone;

methyl glyoxal bis-(phenylhydrazine)hydrazone;

methyl glyoxal bis-(dimethyl 2-hydrazinobenzoate)hydrazone;

methyl glyoxal bis-(4-hydrazinobenzoic acid)hydrazone;

methyl glyoxal bis-(dimethyl 4-hydrazinobenzoate)hydrazone;

methyl glyoxal bis-(2-pyridyl)hydrazone;

methyl glyoxal bis-(diethylene glycol methylether-2-hydrazinobenzoate)hydrazone;

methyl glyoxalbis-[1-(2,3-dihydroxypropane)-2-hydrazinebenzoate]hydrazone;

methyl glyoxal bis-[1-(2-hydroxyethane)-2-hydrazinobenzoate]hydrazone;

methyl glyoxalbis-[(1-hydroxymethyl-1-acetoxy))-2-hydrazino-2-benzoate]hydrazone;

methyl glyoxal bis-[(4-nitrophenyl)-2-hydrazinobenzoate]hydrazone;

methyl glyoxal bis-[(4-methylpyridyl)-2-hydrazinobenzoate]hydrazone;

methyl glyoxal bis-(triethylene glycol 2-hydrazinobenzoate)hydrazone;and

methyl glyoxalbis-(2-hydroxyethylphosphate-2-hydrazinebenzoate)hydrazone.

The above compounds are capable of inhibiting the formation of advancedglycosylation endproducts on target proteins. The cross-linking of theprotein to form the advanced glycosylation endproduct contributes to theentrapment of other proteins and results in the development in vivo ofconditions such as reduced elasticity and wrinkling of the skin, certainkidney diseases, atherosclerosis, osteoarthritis and the like.Similarly, plant material that undergoes nonenzymatic browningdeteriorates and, in the case of foodstuffs, become spoiled or toughenedand, consequently, inedible. Thus, the compounds employed in accordancewith this invention inhibit this late-stage Maillard effect andintervene in the deleterious changes described above.

The rationale of the present invention is to use agents which block thepost-glycosylation step, i.e., the formation of fluorescentchromophores, the presence of which chromophores is associated with, andleads to adverse sequelae of diabetes and aging. An ideal agent wouldprevent the formation of the chromophore and its associate cross-linksof proteins to proteins and trapping of proteins on the other proteins,such as occurs in arteries and in the kidney.

The chemical nature of the early glycosylation products with which thecompounds of the present invention are believed to react may vary, andaccordingly the term "early glycosylation product(s)" as used herein isintended to include any and all such variations within its scope. Forexample, early glycosylation products with carbonyl moieties that areinvolved in the formation of advanced glycosylation endproducts, andthat may be blocked by reaction with the compounds of the presentinvention, have been postulated. In one embodiment, it is envisionedthat the early glycosylation product may comprise the reactive carbonylmoieties of Amadori products or their further condensation, dehydrationand/or rearrangement products, which may condense to form advancedglycosylation endproducts. In another scenario, reactive carbonylcompounds, containing one or more carbonyl moieties (such asglycolaldehyde, glyceraldehyde or 3-deoxyglucosone) may form from thecleavage of Amadori or other early glycosylation endproducts, and bysubsequent reactions with an amine or Amadori product, may form carbonylcontaining advanced glycosylation products such asalkylformyl-glycosylpyrroles.

Several investigators have studied the mechanism of advancedglycosylation product formation. In vitro studies by Eble et al.,(1983), "Nonenzymatic Glucosylation and Glucose-dependent Cross-linkingof Protein", J. Biol. Chem., 258:9406-9412, concerned the cross-linkingof glycosylated protein with nonglycosylated protein in the absence ofglucose. Eble et al. sought to elucidate the mechanism of the Maillardreaction and accordingly conducted controlled initial glycosylation ofRNAase as a model system, which was then examined under varyingconditions. In one aspect, the glycosylated protein material wasisolated and placed in a glucose-free environment and thereby observedto determine the extent of cross-linking.

Eble et al. thereby observed that cross-linking continued to occur notonly with the glycosylated protein but with non-glycosylated proteins aswell. One of the observations noted by Eble et al. was that the reactionbetween glycosylated protein and the protein material appeared to occurat the location on the protein chain of the amino acid lysine.Confirmatory experimentation conducted by Eble et al. in this connectiondemonstrated that free lysine would compete with the lysine on RNAasefor the binding of glycosylated protein. Thus, it might be inferred fromthese data that lysine may serve as an inhibitor of advancedglycosylation; however, this conclusion and the underlying observationsleading to it should be taken in the relatively limited context of themodel system prepared and examined by Eble et al. Clearly, Eble et al.does not appreciate, nor is there a suggestion therein, of thediscoveries that underlie the present invention, with respect to theinhibition of advanced glycosylation of proteins both in vitro and invivo.

The experiments of Eble et al. do not suggest the reactive cleavageproduct mechanism or any other mechanism in the in vivo formation ofadvanced glycosylation endproducts in which glucose is always present.In fact, other investigators support this mechanism to explain theformation of advanced glycosylated endproducts in vivo (see for exampleHayase et al, J. Biol. Chem., 263, pp. 3758-3764 (1989); Sell andMonnier, J. Biol. Chem. 264, pp. 21597-21602 (1989); Oimomi et al.,Agric. Biol. Chem., 53(6):1727-1728 (1989); and Diabetes Research andClinical Practice, 6:311-313 (1989). Accordingly, the use of lysine asan inhibitor in the Eble et al. model system has no bearing upon theutility of the compounds of the present invention in the inhibition ofadvanced glycosylated endproducts formation in the presence of glucosein vivo, and the amelioration of complications of diabetes and aging.

The compositions useful in the present invention comprise or containagents capable of reacting with the active carbonyl intermediate of anearly glycosylation product. Suitable agents are the compounds ofFormula I of the present invention.

The present invention likewise relates to methods for inhibiting theformation of advanced glycosylation endproducts, which comprisecontacting the target proteins with a composition of the presentinvention. In the instance where the target proteins are contained infoodstuffs, whether of plant or animal origin, these foodstuffs couldhave applied to them by various conventional means a compositioncontaining the present agents.

In the food industry, sulfites were found years ago to inhibit theMaillard reaction and are commonly used in processed and stored foods.Recently, however, sulfites in food have been implicated in severe andeven fatal reactions in asthmatics. As a consequence, the sulfitetreatment of fresh fruits and vegetables has been banned. The mechanismfor the allergic reaction is not known.

Accordingly, the present compositions and agents offer a nontoxicalternative to sulfites in the treatment of foods in this manner.

As is apparent from a discussion of the environment of the presentinvention, the present methods and compositions hold the promise forarresting the aging of key proteins both in animals and plants, andconcomitantly, conferring both economic and medical benefits as a resultthereof. In the instance of foodstuffs, the administration of thepresent composition holds the promise for retarding food spoilagethereby making foodstuffs of increased shelf life and greateravailability to consumers. Replacement of currently-used preservatives,such as sulfur dioxide known to cause allergies and asthma in humans,with non-toxic, biocompatible compounds is a further advantage of thepresent invention.

The therapeutic implications of the present invention relate to thearrest of the aging process which has, as indicated earlier, beenidentified in the aging of key proteins by advanced glycosylation andcross-linking. Thus, body proteins, and particularly structural bodyproteins, such as collagen, elastin, lens proteins, nerve proteins,kidney glomerular basement membranes and other extravascular matrixcomponents would all benefit in their longevity and operation from thepractice of the present invention. The present invention thus reducesthe incidence of pathologies involving the entrapment of proteins bycross-linked target proteins, such as retinopathy, cataracts, diabetickidney disease, glomerulosclerosis, peripheral vascular disease,arteriosclerosis obliterans, peripheral neuropathy, stroke,hypertension, atherosclerosis, osteoarthritis, periarticular rigidity,loss of elasticity and wrinkling of skin, stiffening of joints,glomerulonephritis, etc. Likewise, all of these conditions are inevidence in patients afflicted with diabetes mellitus. Thus, the presenttherapeutic method is relevant to treatment of the noted conditions inpatients either of advanced age or those suffering from one of thementioned pathologies.

Protein cross-linking through advanced glycosylation product formationcan decrease solubility of structural proteins such as collagen invessel walls and can also trap serum proteins, such as lipoproteins tothe collagen. Also, this may result in increased permeability of theendothelium and consequently covalent trapping of extravasated plasmaproteins in subendothelial matrix, and reduction in susceptibility ofboth plasma and matrix proteins to physiologic degradation by enzymes.For these reasons, the progressive occlusion of diabetic vessels inducedby chronic hyperglycemia has been hypothesized to result from excessiveformation of glucose-derived cross-links. Such diabetic microvascularchanges and microvascular occlusion can be effectively prevented bychemical inhibition of advanced glycosylation product formationutilizing a composition and the methods of the present invention.

Studies indicate that the development of chronic diabetic damage intarget organs is primarily linked to hyperglycemia so that tightmetabolic control would delay or even prevent end-organ damage. SeeNicholls et al., Lab. Invest., 60, No. 4, p. 486 (1989), which discussesthe effects of islet isografting and aminoguanidine in murine diabeticnephropathy. These studies further evidence that aminoguanidinediminishes aortic wall protein cross-linking in diabetic rats andconfirm earlier studies by Brownlee et al., Science, 232, pp. 1629-1632(1986) to this additional target organ of complication of diabetes.Also, an additional study showed the reduction of immunoglobulintrapping in the kidney by aminoguanidine (Brownlee et al., Diabetes, 35,Suppl. 1, p. 42A (1986)).

Further evidence in the streptozotocin-diabetic rat model thataminoguanidine administration intervenes in the development of diabeticnephropathy was presented by Brownlee et al., 1988, supra, with regardto morphologic changes in the kidney which are hallmarks of diabeticrenal disease. These investigators reported that the increasedglomerular basement membrane thickness, a major structural abnormalitycharacteristic of diabetic renal disease, was prevented withaminoguanidine.

Taken together, these data strongly suggest that inhibition of theformation of advanced glycosylation endproducts (AGEs), by the teachingof the present invention, may prevent late, as well as early, structurallesions due to diabetes, as well as changes during aging caused by theformation of AGEs.

Diabetes-induced changes in the deformability of red blood cells,leading to more rigid cell membranes, is another manifestation ofcross-linking and aminoguanidine has been shown to prevent it in vivo.In such studies, New Zealand White rabbits, with induced, long-termdiabetes are used to study the effects of a test compound on red bloodcell (RBC) deformability (df). The test compound is administered at arate of 100 mg/kg by oral garage to diabetic rabbits.

A further consequence of diabetes is the hyperglycemia-induced matrixbone differentiation resulting in decreased bone formation usuallyassociated with chronic diabetes. In animal models, diabetes reducesmatrix-induced bone differentiation by 70%.

In the instance where the compositions of the present invention areutilized for in vivo or therapeutic purposes, it may be noted that thecompounds or agents used therein are biocompatible. Pharmaceuticalcompositions may be prepared with a therapeutically effective quantityof the agents or compounds of the present invention and may include apharmaceutically acceptable carrier, selected from known materialsutilized for this purpose. Such compositions may be prepared in avariety of forms, depending on the method of administration. Also,various pharmaceutically acceptable addition salts of the compounds ofFormula I may be utilized.

A liquid form would be utilized in the instance where administration isby intravenous, intramuscular or intraperitoneal injection. Whenappropriate, solid dosage forms such as tablets, capsules, or liquiddosage formulations such as solutions and suspensions, etc., may beprepared for oral administration. For topical or dermal application tothe skin or eye, a solution, a lotion or ointment may be formulated withthe agent in a suitable vehicle such as water, ethanol, propyleneglycol, perhaps including a carrier to aid in penetration into the skinor eye. For example, a topical preparation could include up to about 10%of the compound of Formula I. Other suitable forms for administration toother body tissues are also contemplated.

In the instance where the present method has therapeutic application,the animal host intended for treatment may have administered to it aquantity of one or more of the agents, in a suitable pharmaceuticalform. Administration may be accomplished by known techniques, such asoral, topical and parenteral techniques such as intradermal,subcutaneous, intravenous or intraperitoneal injection, as well as byother conventional means.

Administration of the agents may take place over an extended period oftime at a dosage level of, for example, up to about 30 mg/kg.

As noted earlier, the invention also extends to a method of inhibitingthe discoloration of teeth resulting from nonenzymatic browning in theoral cavity which comprises administration to a subject in need of suchtherapy an amount effective to inhibit the formation of advancedglycosylation endproducts of a composition comprising an agent ofstructural Formula I.

The nonenzymatic browning reaction which occurs in the oral cavityresults in the discoloration of teeth. Presently used anti-plaque agentsaccelerate this nonenzymatic browning reaction and further the stainingof the teeth. Recently, a class of cationic anti-microbial agents withremarkable anti-plaque properties have been formulated in oral rinsesfor regular use to kill bacteria in the mouth. These agents, thecationic antiseptics, include such agents as alexidine, cetyl pyridiniumchloride, chlorhexidine gluconate, hexetidine, and benzalkoniumchloride.

Tooth staining by chlorhexidine and other anti-plaque agents apparentlyresults from the enhancement of the Maillard reaction. Nordbo, J. Dent.Res., 58, p. 1429 (1979) reported that chlorhexidine and benzalkoniumchloride catalyze browning reactions in vitro. Chlorhexidine added tomixtures containing a sugar derivative and a source of amino groupsunderwent increased color formation, attributed to the Maillardreaction. It is also known that use of chlorhexidine results in anincreased dental pellicle. Nordbo proposed that chlorhexidine resultedin tooth staining in two ways: first, by increasing formation ofpellicle which contains more amino groups, and secondly, by catalysis ofthe Maillard reaction leading to colored products.

In accordance with this method, the compounds of Formula I areformulated into compositions adapted for use in the oral cavity.Particularly suitable formulations are oral rinses and toothpastesincorporating the active agent.

In the practice of this invention, conventional formulating techniquesare utilized with nontoxic, pharmaceutically acceptable carrierstypically utilized in the amounts and combinations that are well-knownfor the formulation of such oral rinses and toothpastes.

The agent of Formula I is formulated in compositions in an amounteffective to inhibit the formation of advanced glycosylationendproducts. This amount will, of course, vary with the particular agentbeing utilized and the particular dosage form, but typically is in therange of 0.01% to 1.0%, by weight, of the particular formulation.

The compounds encompassed by Formula I are novel compounds which can beprepared by modifications of chemical syntheses well-known in the art.

The compounds of formula I can be prepared according to the methodsdescribed in Oliverio et al., J. Pharm. Sci., 52:2, pp. 202-203 (1963)or as shown in the various schemes below. ##STR6##

In this reaction Scheme, the appropriate dioxo compound of formula IIwherein W, R₁, and R₂ are as hereinbefore defined, is reacted with theappropriate salt of a hydrazine of formula III wherein R' is a pyridyl,phenyl, carboxyphenyl, or alkoxycarbonylphenyl group, to afford thedesired compound of formula I wherein W, R₁, R₂ and R' are ashereinbefore defined.

Typically, this reaction is conducted at the reflux temperature of thereaction solvent. for times of 1-24 hours. Typically, a polar solventsuch as ethanol is utilized for the conduct of the reaction.

The compounds of formula I wherein the R group is a water solubilizingester moiety are typically prepared from the corresponding compound offormula I wherein R is hydrogen or the sodium salt thereof. Typically,the sodium salt of the carboxy group is prepared by reaction of thecompound of formula I wherein R is a carboxy group with sodiumhydroxide. This sodium salt can then be used to prepare various estersby condensing the sodium salt with an alcohol, glycol or haloalkylcompound to afford the desired ester. The preparation of the phosphoricacid esters involves treatment of a compound of formula I having a freecarboxy or hydroxy group with phosphorus oxychloride and trimethylphosphate to afford the desired ester.

The following examples are illustrative of the invention.

EXAMPLE 1 Methylglyoxal Bis-(2-Hydrazinobenzoic Acid) Hydrazone

To a solution of 2-hydrazinobenzoic acid hydrochloride (3.0 g, 15.9mmol) in water (5 mL) was added slowly the methyl glyoxal (40% aqueous,1.43 mL, 7.93 mmol) in ethyl alcohol (25 mL). The solution was refluxedfor 3 hours and stirred at room temperature for 2 hours and then kept at-20° C. overnight. The yellow product which separated was filtered,washed with ethyl alcohol and dried. Crystallization fromdimethylformamide affords methyl glyoxal bis-(2-hydrazinobenzoic acid)hydrazone in 83% yield (2.24 g), mp. 297°-299° C. (dec).

EXAMPLE 2 Methylglyoxal Bis-(2-Hydrazinobenzoic Acid) Hydrazone (Nasalt)

To a solution containing sodium hydroxide (2.40 g) in water (100 mL) wasadded methylglyoxal bis-(2-hydrazinobenzoic acid) hydrazone (10.0 g,0.02 mol) in one portion and the reaction stirred for 30 minutes at roomtemperature. The pH was checked to make sure the solution was basic andthe reaction was evaporated to dryness under reduced pressure withheating to afford 11.10 g (100%) of the sodium salt of ethylglyoxalbis-(2-hydrazinobenzoic acid) hydrazone as an orange solid, mp.339°-341° C.

EXAMPLE 3 Methyl Glyoxal Bis-(Dimethyl 2-Hydrazinobenzoate)Hydrazone

To a solid sample of methylglyoxal bis-(2-hydrazinobenzoic acid)hydrazone (200 mg, 0.57 mmol) was added dropwise a freshly preparedsolution of diazomethane (in ether) with stirring until the evolution ofnitrogen ceased and yellow color persisted. The reaction was monitoredby thin layer chromatography using ethyl acetate/hexane 1:1 as asolvent. When the reaction was complete, the solution was evaporated todryness to yield 200 mg (95%) of methyl glyoxal bis-(dimethyl2-hydrazinobenzoate) hydrazone, mp. 170°-173° C.

EXAMPLE 4 Methyl Glyoxal Bis-(Phenylhydrazine) Hydrazone

To phenyl hydrazine (7.16 g, 66.3 mmol) was added slowly methyl gloxal(40% aqueous, 3.99 g, 22 mmol) in ethyl alcohol (88 mL). The solutionwas refluxed for 19 hours and then cooled to room temperature. Theyellow product which separated was filtered, washed with ethyl alcoholand dried to give methyl glyoxal bis-(phenylhydrazine) hydrazone 5.02 g(32%), mp. 121°-124° C.

EXAMPLE 5 Methyl Glyoxal Bis-(Diethyl 2-Hydrazinobenzoate) Hydrazone

To ethyl 2-hydrazinobenzoate (5.96 g, 33 mmol) was slowly added methylglyoxal (40% aqueous, 2.0 g, 11 mmol) in ethyl alcohol (44 mL). Thesolution was refluxed for 24 hours and cooled to room temperature. Theyellow product which separated was filtered, washed with ethyl alcoholand dried to give methyl glyoxal bis-(diethyl 2-hydrazinobenzoate)hydrazone 4.15 g (95%), mp. 133°-135° C.

EXAMPLE 6 Methyl Glyoxal Bis-(4-Hydrazinobenzoic Acid) Hydrazone

To 4-hydrazinobenzoic acid (5.04 g, 33 mmol) was slowly added methylglyoxal (40% aqueous, 2.0 g, 11 mmol) in ethyl alcohol (44 mL). Thesolution was refluxed for 18 hours and cooled to room temperature. Theyellow product which separated was filtered, washed with ethyl alcoholand dried. Crystallization from dimethylformamide affords methyl glyoxalbis-(4-hydrazinobenzoic acid) hydrazone, 4.20 g (37%), mp. 339°-340° C.

EXAMPLE 7 Methyl Glyoxal Bis-(Dimethyl 4-Hydrazinobenzoate) Hydrazone

To a solid sample of methyl glyoxal his-(4-hydrazinobenzoic acid)hydrazone (206 mg, 0.59 mmol) was added dropwise a freshly preparedsolution of diazomethane (in ether) with stirring until the evolution ofnitrogen ceased and yellow color persisted. The reaction was monitoredby thin layer chromatography using ethyl acetate/hexane 1:1 as asolvent. When the reaction was complete, the solution was evaporated todryness to yield 200 mg (93%), of the title compound, mp. 268°-270° C.

EXAMPLE 8 Methyl Glyoxal Bis-(2-Pyridyl) Hydrazone

To 2-hydrazinopyridine (4.36 g, 40 mmol) was added slowly methyl glyoxal(40% aqueous, 3.99 g, 22 mmol) in ethyl alcohol (88 mL). The solutionwas refluxed for 17 hours and cooled to room temperature, The yellowproduct which separated was filtered, washed with ethyl alcohol anddried to give methyl glyoxal bis-(2-pyridyl) hydrazone 3.00 g (29%), mp.196°-198° C. as the free base. The entire product was added to water andhydrogen chloride gas was bubbled through the solution until thesolution was clear. The water was evaporated to give the hydrochloridesalt, mp. 270°-270° C.

EXAMPLE 9 Methyl Glyoxal Bis-(Diethylene Glycol Methyl Ether2-Hydrazinobenzoate) Hydrazone

To a solution of the sodium salt of methylglyoxalbis-(2-hydrazinobenzoic acid) hydrazone (807 mg, 2.1 mmol) indimethylformamide (16 mL) was added 1-bromo-2-(2-methoxyethoxy) ethane(780 mg, 4.2 mmol) at 80° C. for 5.5 hours. The reaction was cooled andpartitioned between ethyl acetate and water and the organic layer wasexhaustively washed with water. The ethyl acetate layer was dried andevaporated to give 800 mg (70%) of methyl glyoxal bis-(diethylene glycolmethyl ether 2-hydrazinobenzoate) hydrazone as a yellow solid, mp.64°-66° C.

EXAMPLE 10 Methyl GlyoxalBis-[1-(2,3-Dihydroxypropane)-2-Hydrazinobenzoate] Hydrazone

To a solution of methylglyoxal bis-(2-hydrazinobenzoic acid) hydrazonesodium salt (3.0 g. 7.8 mmol) in dimethylformamide (35 mL) was added3-bromo-1,2-propanediol (2.55 g, 16.5 mmol) at 80° C. for 17 hours. Thereaction was cooled and partitioned between ethyl acetate and water andthe organic layer was exhaustively washed with water. The ethyl acetatelayer was dried and evaporated to give 2.6 g (68%) of methyl glyoxalbis-[1-(2,3-dihydroxypropane)-2-hydrazinobenzoate] hydrazone as a yellowsolid, mp. 181°-183° C.

EXAMPLE 11 Methyl Glyoxal Bis-[1-(2-Hydroxyethane)-2-Hydrazinobenzoate]Hydrazone

To a solution of the sodium salt of methylglyoxalbis-(2-hydrazinobenzoic acid) hydrazone (3.0 g, 7.81 mmol) indimethylformamide (35 mL) was added 2-bromoethanol (2.25 g, 18.0 mmol)at 80° C. for 3 hours. The reaction was cooled and partitioned betweenethyl acetate and water and the organic layer was exhaustively washedwith water. The ethyl acetate layer was dried and evaporated to give 2.4g (72%) of the title compound, mp. 165°-168° C.

EXAMPLE 12 Methyl GlyoxalBis-[(1-Hydroxymethyl-1-Acetoxy)-2-Hydrazinobenzoate] Hydrazone

To a solution of the sodium salt of methylglyoxalbis-(2-hydrazinobenzoic acid) hydrazone (628 mg, 1.63 mmol) indimethylformamide (8 mL) was added bromomethyl acetate (540 mg, 3.53mmol) at 80° C. for 17.5 hours. The reaction was filtered and dried togive 100 mg (15%) of the title compound, mp. 155°-157° C.

EXAMPLE 13 Methyl Glyoxal Bis-[4-Nitrophenyl)-2-Hydrazinobenzoate]Hydrazone

To a solution of the sodium salt of methylglyoxalbis-(2-hydrazinobenzoic acid) hydrazone (100 mg, 0.26 mmol) indimethylformamide (2 mL) was added 4-nitrobenzyl bromide (113 mg, 0.52mmol) at 80° C. for 17.5 hours. The reaction was filtered and dried togive 100 mg (63%) of the title compound, as a yellow solid, mp.264°-266° C.

EXAMPLE 14 Methyl Glyoxal Bis-[4-Methylpyridyl)-2-Hydrazinobenzoate]Hydrazone

To a solution of the sodium salt of methylglyoxalbis-(2-hydrazinobenzoic acid) hydrazone (200 mg, 0.52 mmol) indimethylformamide (6 mL) and sodium hydride (36 mg, 1.5 mmol) was added4-picoloyl chloride hydrochloride (171 mg, 1.0 mmol) at 80° C. for 18hours. The reaction was cooled and partitioned between ethyl acetate andwater and the organic layer as exhaustively washed with water. The ethylacetate layer was dried and evaporated to give 150 mg (55%) of methylglyoxal bis-[4-methylpyridyl)-2-hydrazinobenzoate] hydrazone as a yellowsolid, mp. >350° C.

EXAMPLE 15 Methyl Glyoxal Bis-(Triethylene Glycol 2-Hydrazinobenzoate)Hydrazone

To a solution of methylglyoxal bis-(2-hydrazino-benzoic acid) hydrazonesodium salt (3.0 g, 7.8 mmol) in dimethylformamide (35 mL) was added2-[2-(2-chloroethyloxy)ethoxy]ethanol(2.88 g, 17.0 mmol) at 80° C. for17 hours. The reaction mixture was cooled, partitioned between ethylacetate and water, and the organic layer exhaustively washed with water.The ethyl acetate layer was evaporated to dryness, and the residuerecrystallized from acetone to give 3.1 g (66%) of methyl glyoxalbis-(triethylene glycol 2-hydrazinobenzoate) hydrazone as a yellowsolid, mp. 59°-62° C.

EXAMPLE 16 Methyl GlyoxalBis-(2-Hydroxyethylphosphate-2-Hydrazinobenzoate) Hydrazone

To a solution of phosphorus oxychloride (80 μL, 0.86 mmol) and trimethylphosphate (2.07 mL, 17.7 mmol) at 0° C. for 30 minutes was added methylglyoxal bis-[1-(2-hydroxyethane)-2-hydrazinobenzoate] hydrazone (207 mg,0.48 mmol) at 0° C. for 4 hours whereby the solution became homogenous.Water (2 mL) was added, followed by ammonium hydroxide until thesolution became neutral, and refluxed for 30 minutes. The reaction wascooled to room temperature and the solid filtered, yielding 200 mg (71%)of methyl glyoxal bis-(2-hydroxyethylphosphate-2-hydrazinobenzoate)hydrazone, mp. 197°-200° C. (dec).

EXAMPLE 17

The following method was used to evaluate the ability of the compoundsof the present invention to inhibit the cross-linking of glycated bovineserum albumin (AGE-BSA), to the rat tail tendon collagen coated 96-wellplate.

The AGE-BSA was prepared by incubating BSA at a concentration of 200 mgper ml with 200 mM glucose in 0.4M sodium phosphate buffer, pH 7.4 at37° C. for 12 weeks. The glycated BSA was then extensively dialyzedagainst phosphate buffer solution (PAS) for 48 hours with additional 5times buffer exchanges. The rat tail tendon collagen coated plate wasblocked first with 300 μl of superbloc blocking buffer (Pierce #37515X)for one hour. The blocking solution was removed from the wells bywashing the plate twice with PAS-Tween 20 solution (0.05% Tween 20)using a NUNC-multiprobe or Dynatech ELISA-plate washer. Cross-linking ofAGE-BSA (1 to 10 μg per well depending on the batch of AGE-BSA) to rattail tendon collagen coated plate was performed with and without thetesting compound dissolved in PAS buffer at pH 7.4 at the desiredconcentrations by the addition of 50 μl each of the AGE-BSA diluted inPAS or in the testing compound at 37° C. for 4 hours. The unbrowned BSAin PAS buffer with or without testing compound were added to theseparate wells as the blanks. The un-cross-linked AGE-BSA was thenremoved by washing the wells three times with PAS-Tween buffer. Thecross-linked AGE-BSA to the tail tendon coated plate was thenquantitated by the polyclonal antibody raised against AGE-RNase. After aone-hour incubation period, AGE antibody was removed by washing 4 timeswith PAS-Tween.

The bound AGE antibody was then detected with the addition ofhorseradish peroxidase-conjugated secondary antibody, e.g., goatanti-rabbit immunoglobulin and incubation for 30 minutes. The substrateof 2,2-azino-di(3-ethylbenzthiazoline sulfonic acid) (ABTS chromogen)(Zymed #00-2011) was added. The reaction was allowed for an additional15 minutes and the absorbance was read at 410 nm in a Dynatech platereader.

The % inhibition of each test compound was calculated as follows.

    % inhibition={[Optical density (without compound)-optical density (with compound)]/optical density (without compound)]}100%

The IC₅₀ relative inhibition by various test compounds at 10 mM is asfollows:

    ______________________________________                                        Test Compound           IC.sub.50                                             ______________________________________                                        methyl glyoxal bis(2-hydrazino-                                                                       0.04   mM                                             benzoic acid)hydrazone                                                        methyl glyoxal bis(4-hydrazinobenzoic                                                                 11.48  mM                                             acid)hydrazone                                                                methyl glyoxal bis-(dimethyl                                                                          10     mM                                             hydrazinobenzoate)hydrazone                                                   ______________________________________                                    

The above experiment suggests that this type of drug therapy hasbenefits in reducing the pathology associated with the advancedglycosylation of proteins and the formation of cross-links betweenproteins and other macromolecules. Drug therapy may be used to preventthe increased trapping and cross-linking of proteins that occurs indiabetes and aging which leads to sequelae such as retinal damage, andextra-vascularly, damage to tendons, ligaments and other joints.

EXAMPLE 18

The following method was used to evaluate the ability of the compoundsof the present invention to inhibit the cross-linking of N-acetylglycyl-lysine methyl ester in the presence of ribose.

Materials:

N-acetylglycyllysine methyl ester (DP in formula below) Ribose (R informula below)

Test compounds (C in formula below)

Reagents:

0.5M sodium phosphate buffer pH 7.4 N-acetylglycyllysine methyl ester in0.5M sodium phosphate buffer, pH 7.4

Ribose: 800 mM

Test compounds dissolved in the above buffer and the pH is adjusted to7.4, if necessary

Procedure:

Reaction mixtures are prepared as follows:

    ______________________________________                                        80 mg/ml N-acetylglycyllysine                                                                    0.1       0.1   --                                         methyl ester/buffer                                                           ribose             0.1       0.1   0.1                                        test compound      --        0.1   0.1                                        buffer             0.2       0.1   0.2                                        ______________________________________                                    

and incubated at 37 C for 16-24 hours. At the end of the incubationperiod, the fluroescence is read using an excitation wavelength of 350nm and emission wavelenth of 400 nm. The inhibition of the cross-linkingis calculated from the decrease in the fluorescence in the presence ofthe test compounds according to the formula:

    Inhibition (%)=100×[DPRC fluorescence-RC fluorescence]/DPR fluorescence

The Inhibition by various test compounds (IC₅₀) is as follows:

    ______________________________________                                        Test compound          IC.sub.50 mM                                           ______________________________________                                        methyl glyoxal bis-(2-hydrazino-                                                                     0.156                                                  benzoic acid)hydrazone                                                        methyl glyoxal bis-(dimethyl 2-                                                                      >10                                                    hydrazinobenzoate)hydrazone                                                   methyl glyoxal bis-(phenyl-                                                                          >10                                                    hydrazine)hydrazone                                                           methyl glyoxal bis-(diethyl 2-                                                                       10                                                     hydrazinobenzoate)hydrazone                                                   methyl glyoxal bis-(4-hydrazino-                                                                     0.118                                                  benzoic acid)hydrazone                                                        methyl glyoxal bis-(dimethyl 4-                                                                      10                                                     hydrazinobenzoate)hydrazone                                                   methyl glyoxal bis-(2-pyridyl)hydrazone                                                              3.912                                                  methyl glyoxal bis-(diethylene glycol                                                                >10                                                    methyl ether-2-hydrazinobenzoate)hydrazone                                    methyl glyoxal bis-[1-(2,3-dihydroxy-                                                                >3                                                     propane)-2-hydrazinobenzoate]hydrazone                                        methyl glyoxal bis-[1-(2-hydroxyethane)-                                                             3                                                      2-hydrazinobenzoate]hydrazone;                                                methyl glyoxal bis-[(l-hydroxympthyl-1-                                                              0.764                                                  acetoxy))-2-hydrazino-2-benzoate]hydrazone                                    methyl glyoxal bis-[(4-nitrophenyl)-                                                                 10                                                     2-hydrazinobenzoate]hydrazone                                                 methyl glyoxal bis-[(4-methylpyridyl)-2-                                                             1.24                                                   hydrazinobenzoate]hydrazone                                                   methyl glyoxal bis-(triethylene glycol 2-                                                            1.53                                                   hydrazinobenzoate)hydrazone                                                   methyl glyoxal bis-(2-hydroxyethylphos-                                                              0.597                                                  phate-2-hydrazinebenzoate)hydrazone                                           ______________________________________                                    

The above experiment suggests that this type of drug therapy will reducethe pathology associated with the advanced glycosylation of proteins andthe formation of cross-links between proteins and other macromolecules.Drug therapy may be used to prevent the increased trapping andcross-linking of proteins that occurs in diabetes and aging which leadsto sequelae such as retinal damage, and extra-vascularly, damage totendons, ligaments and other joints.

EXAMPLE 19

    ______________________________________                                        Tablet            mg/tablet                                                   ______________________________________                                        Compound of Formula I                                                                           50                                                          Starch            50                                                          Mannitol          75                                                          Magnesium stearate                                                                              2                                                           Stearic acid      5                                                           ______________________________________                                    

The compound, a portion of the starch and the lactose are combined andwet granulated with starch paste. The wet granulation is placed on traysand allowed to dry overnight at a temperature of 45° C. The driedgranulation is comminuted in a comminutor to a particle size ofapproximately 20 mesh. Magnesium stearate, stearic acid and the balanceof the starch are added and the entire mix blended prior to compressionon a suitable tablet press. The tablets are compressed at a weight of232 mg. using a 1 1/32" punch with a hardness of 4 kg. These tabletswill disintegrate within a half hour according to the method describedin USP XVI.

EXAMPLE 20

    ______________________________________                                        Lotion               mg/g                                                     ______________________________________                                        Compound of Formula I                                                                              1.0                                                      Ethyl alcohol        400.0                                                    Polyethylene glycol 400                                                                            300.0                                                    Hydroxypropyl cellulose                                                                            5.0                                                      Propylene glycol     to make 1.0                                                                             g                                              ______________________________________                                    

EXAMPLE 21

    ______________________________________                                        Oral Rinse                                                                    ______________________________________                                        Compound of Formula I:                                                                           1.4%                                                       Chlorhexidine-gluconate                                                                         0.12%                                                       Ethanol           11.6%                                                       Sodium saccharin  0.15%                                                       FD&C Blue No. 1   0.001%                                                      Peppermint Oil     0.5%                                                       Glycerine         10.0%                                                       Tween 60           0.3%                                                       Water to           100%                                                       ______________________________________                                    

EXAMPLE 22

    ______________________________________                                        Toothpaste                                                                    ______________________________________                                        Compound of Formula I:                                                                              5.5%                                                    Sorbitol, 70% in water                                                                               25%                                                    Sodium saccharin     0.15%                                                    Sodium lauryl sulfate                                                                              1.75%                                                    Carbopol 934, 6% dispersion in                                                                       15%                                                    Oil of Spearmint      1.0%                                                    Sodium hydroxide, 50% in water                                                                     0.76%                                                    Dibasic calcium phosphate dihydrate                                                                  45%                                                    Water to              100%                                                    ______________________________________                                    

EXAMPLE 23

To further study the ability of inhibitors of nonenzymatic browning toprevent the discoloration of protein on a surface, such as that whichoccurs on the tooth surface, the following surface browning experimentis performed. As a substitute for a pellicle-covered tooth surface,unexposed and developed photographic paper is used to provide a fixedprotein (gelatin, i.e., collagen) surface on a paper backing. Fivemillimeter circles are punched and immersed for one week at 50° C. in asolution of 100 Mm glucose-6-phosphate in a 0.5M phosphate buffer, pH7.4, containing 3 Mm sodium azide. Glucose-6-phosphate is a sugarcapable of participating in nonenzymatic browning at a more rapid ratethan glucose. In addition to the glucose-6-phosphate, chlorhexidineand/or a compound of Formula I are included. After incubation, thegelatin/paper disks are rinsed with water, observed for brown color, andphotographed.

Incubation of the disks in glucose-6-phosphate alone shows slight browncolor versus disks soaked in buffer alone. Inclusion of chlorhexidine(in the form of Peridex® at a final concentration of 0.04%chlorhexidine) shows significant browning. Addition of a compound ofFormula I to the chlorhexidine completely inhibits browning of thegelatin, as does inclusion of a compound of Formula I in the absence ofchlorhexidine.

The slight brown color formed by the action of glucose-6-phosphate onthe gelatin surface alone and its prevention by a compound of Formula Idemonstrates the utility of the present invention in preventingnonenzymatic browning of tooth surfaces. The enhanced browning in thepresence of chlorhexidine and its prevention with a compound of FormulaI demonstrates the utility of the present invention in preventing theanti-plaque agent-enhanced nonenzymatic browning which occurs withchlorhexidine.

This invention maybe embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allrespects illustrative and not restrictive, the scope of the inventionbeing indicated by the appended claims, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

What is claimed is:
 1. A compound of the formula ##STR7## wherein R is acarboxylic acid substituted phenyl group of the formula ##STR8## whereinR is hydrogen, lower alkyl or a water-solubilizing ester moiety; W is acarbon-carbon bond;R₁ is a methyl group; R₂ is hydrogen;and theirbiologically or pharmaceutically acceptable acid addition salts.
 2. Thecompound of claim 1 which is methyl glyoxal bis-(2-hydrazinobenzoicacid)hydrazone hemihydrate, or another pharmaceutically acceptable saltthereof.
 3. The compound of claim 1 which is methyl glyoxalbis-(4-hydrazinobenzoic acid)hydrazone, or a pharmaceutically acceptablesalt thereof.
 4. The compound of claim 1 which is methyl glyoxalbis-[1-(2,3-dihydroxypropane)-2-hydrazinebenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 5. The compound of claim 1which is methyl glyoxalbis-[1-(2-hydroxyethane)-2-hydrazinobenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 6. The compound of claim 1which is methyl glyoxalbis-[(1-hydroxymethyl-1-acetoxy))-2-hydrazino-2-benzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 7. The compound of claim 1which is methyl glyoxalbis-[(4-methylpyridyl)-2-hydrazinobenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 8. The compound of claim 1which is methyl glyoxal bis-(triethylene glycol2-hydrazinobenzoate)hydrazone, or a pharmaceutically acceptable saltthereof.
 9. The compound of claim 1 which is methyl glyoxalbis-(2-hydroxyethylphosphate-2-hydrazinebenzoate)hydrazone, or apharmaceutically acceptable salt thereof.
 10. A composition forinhibiting the advanced glycosylation of a target protein comprising aneffective amount of a compound of claim 1 together with a carriertherefor.
 11. A pharmaceutical composition for administration to ananimal to inhibit the advanced glycosylation of a target protein withinsaid animal, comprising a pharmaceutically effective amount of acompound of the formula ##STR9## wherein R is a pyridyl, phenyl or acarboxylic acid substituted phenyl group of the formula ##STR10##wherein R is hydrogen, lower alkyl or a water-solubilizing ester moiety;W is a carbon-carbon bond or an alkylene group of 1-3 carbon atoms, andR₁ is a lower alkyl, aryl, or heteroaryl group and R₂ is hydrogen, alower alkyl, aryl or heteroaryl group;and their biologically orpharmaceutically acceptable acid addition salts, together with a carriertherefor.
 12. The composition of claim 11 wherein W is a carbon-carbonbond, R₁ is a lower alkyl group, and R₂ is hydrogen.
 13. The compositionof claim 12 which is methyl glyoxal bis-(2-hydrazinobenzoicacid)hydrazone hemihydrate, or another pharmaceutically acceptable saltthereof.
 14. The composition of claim 12 which is methyl glyoxalbis-(4-hydrazinobenzoic acid)hydrazone, or a pharmaceutically acceptablesalt thereof.
 15. The composition of claim 12 which is methyl glyoxalbis-(2-pyridyl)hydrazone hydrochloride, or another pharmaceuticallyacceptable salt thereof.
 16. The composition of claim 12 which is methylglyoxal bis-[1-(2,3-dihydroxypropane)-2-hydrazinebenzoate]hydrazone, ora pharmaceutically acceptable salt thereof.
 17. The composition of claim12 which is methyl glyoxalbis-[1-(2-hydroxyethane)-2-hydrazinobenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 18. The composition of claim12 which is methyl glyoxalbis-[(1-hydroxymethyl-1-acetoxy))-2-hydrazino-2-benzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 19. The composition of claim12 which is methyl glyoxalbis-[(4-methylpyridyl)-2-hydrazinobenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 20. The composition of claim12 which is methyl glyoxal bis-(triethylene glycol2-hydrazinobenzoate)hydrazone, or a pharmaceutically acceptable saltthereof.
 21. The composition of claim 12 which is methyl glyoxalbis-(2-hydroxyethylphosphate-2-hydrazinebenzoate) hydrazone, or apharmaceutically acceptable salt thereof.
 22. A method for treating ananimal to inhibit the formation of advanced glycosylation endproducts ofa target protein within said animal, said method comprisingadministering an effective amount of a pharmaceutical composition, saidpharmaceutical composition comprising a compound of the formula##STR11## wherein R is a pyridyl, phenyl or a carboxylic acidsubstituted phenyl group of the formula ##STR12## wherein R is hydrogen,lower alkyl or a water-solubilizing ester moiety; W is a carbon-carbonbond or an alkylene group of 1-3 carbon atoms, and R₁ is a lower alkyl,aryl, or heteroaryl group and R₂ is hydrogen, a lower alkyl, aryl orheteroaryl group;and their biologically or pharmaceutically acceptableacid addition salts, together with a carrier therefor.
 23. The method ofclaim 22 wherein W is a carbon-carbon bond, R₁ is a lower alkyl group,and R₂ is hydrogen.
 24. The method of claim 23 which is methyl glyoxalbis-(2-hydrazinobenzoic acid)hydrazone hemihydrate, or anotherpharmaceutically acceptable salt thereof.
 25. The method of claim 23which is methyl glyoxal bis-(4-hydrazinobenzoic acid)hydrazone, or apharmaceutically acceptable salt thereof.
 26. The method of claim 23which is methyl glyoxal bis-(2-pyridyl)hydrazone hydrochloride, oranother pharmaceutically acceptable salt thereof.
 27. The method ofclaim 23 which is methyl glyoxalbis-[1-(2,3-dihydroxypropane)-2-hydrazinebenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 28. The method of claim 23which is methyl glyoxalbis-[1-(2-hydroxyethane)-2-hydrazinobenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 29. The method of claim 23which is methyl glyoxalbis-[(1-hydroxymethyl-1-acetoxy))-2-hydrazino-2-benzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 30. The method of claim 23which is methyl glyoxalbis-[(4-methylpyridyl)-2-hydrazinobenzoate]hydrazone, or apharmaceutically acceptable salt thereof.
 31. The method of claim 23which is methyl glyoxal bis-(triethylene glycol2-hydrazinobenzoate)hydrazone, or a pharmaceutically acceptable saltthereof.
 32. The method of claim 23 which is methyl glyoxalbis-(2-hydroxyethylphosphate-2-hydrazinebenzoate) hydrazone, or apharmaceutically acceptable salt thereof.
 33. A method of inhibiting thediscoloration of teeth resulting from non-enzymatic browning in the oralcavity which comprises administration of an amount effective to inhibitthe formation of advanced glycosylation endproducts of a compositioncomprising a compound of claim 1, together with a carrier therefor.